title: Development of a multiplex sandwich aptamer microarray for the detection of VEGF165 and thrombin.
abstract: In this work we have developed a multiplex microarray system capable of detecting VEGF165 and thrombin. We recently described a Sandwich Aptamer Microarray (SAM) for thrombin detection feasible for use in multiplex microarrays; here we describe a new aptasensor for VEGF165 detection employing Vap7 and VEa5, two DNA aptamers recognizing different sites of the protein. The aptamers were modified to be adapted to the solid phase platform of SAM and their capability to simultaneously recognize VEGF165 by forming a ternary complex was analyzed in solution. Having so defined the best tandem arrangement of modified aptamers, we set up the aptasensor for VEGF165, and finally analyzed the multiplex system with the two aptasensors for the simultaneous detection of VEGF165 and thrombin. The results indicate that each sandwich is specific, even when the two proteins are mixed. The system performance is consistent with the behavior evidenced by the biochemical analysis, which proves to be valuable to drive the evaluation and refinement of aptamers prior to or along the development of a detection platform. Since thrombin upregulates VEGF expression, the simultaneous recognition of these two proteins could be useful in the analysis of biomarkers in pathologies characterized by neo-angiogenesis.
authors.affiliation: Dipartimento di Scienze del Farmaco, Università di Padova, via Marzolo 5, 35131 Padova, Italy. barbara.gatto@unipd.it.
authors.forename: Alice
authors.initials: A
authors.lastname: Sosic
authors.forename: Anna
authors.initials: A
authors.lastname: Meneghello
authors.forename: Agnese
authors.initials: A
authors.lastname: Antognoli
authors.forename: Erica
authors.initials: E
authors.lastname: Cretaio
authors.forename: Barbara
authors.initials: B
authors.lastname: Gatto
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation*; D045424:Complex Mixtures / Q000097:blood*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005240:Feasibility Studies; D006801:Humans; D040081:Protein Array Analysis / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis*; D042461:Vascular Endothelial Growth Factor A / Q000097:blood*
chemical_list: D052157:Aptamers, Nucleotide; D045424:Complex Mixtures; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013917:Thrombin